Method for identifying a biomolecule using a terahertz sensor control system

ABSTRACT

A system for biomolecule identification by terahertz sensing, an asymmetric triple split-rectangular (ATSR) metamaterial biosensor, and a method for biomolecule identification by terahertz sensing are presented. The asymmetric triple split-rectangular (ATSR) metamaterial biosensor includes three gap areas which highly confine an electric field. The biosensor includes an E-shaped structure facing an inverted E-shaped structure with gaps between the respective legs. Each leg has a specially designed extension on either side which increases the electric field. A terahertz laser interrogates an analyte upon the metamaterial structure with a plurality of frequencies. The amplitude difference is estimated by an amplitude difference referencing technique. The amplitude difference is matched to a database record to identify the biomolecule analyte. The asymmetric triple split-rectangular (ATSR) metamaterial biosensor in combination with the amplitude difference referencing technique detects the type of biomolecule with a high degree of accuracy and requires only small analyte samples with sub-micron thicknesses.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a Continuation of U.S. application Ser. No.17/237,205, now allowed, having a filing date of Apr. 22, 2021, which isa Continuation of U.S. application Ser. No. 16/416,926, now U.S. Pat.No. 11,041,802, having a filing date of May 20, 2019.

STATEMENT REGARDING PRIOR DISCLOSURE BY THE INVENTORS

Aspects of this technology are described in an article “Evaluation ofamplitude difference referencing technique with terahertz metasurfacesfor sub-micron analytes sensing” published in Journal of King SaudUniversity—Science, DOI: 10.1016/j.jksus.2018.11.011, on Nov. 28, 2018,which is incorporated herein by reference in its entirety.

BACKGROUND Technical Field

The present disclosure is directed to an asymmetric triplesplit-rectangular (ATSR) metamaterial biosensor for sub-micron thicknessanalyte identification at terahertz frequencies. The frequency responseis post-processed by an amplitude difference referencing technique.

Description of Related Art

The “background” description provided herein is for the purpose ofgenerally presenting the context of the disclosure. Work of thepresently named inventors, to the extent it is described in thisbackground section, as well as aspects of the description which may nototherwise qualify as prior art at the time of filing, are neitherexpressly or impliedly admitted as prior art against the presentinvention.

The past two decades have revealed a plethora of terahertz (THz)applications emerging from several fields of science. (See Jepsen, P.U., Cooke, D. G., Koch, M., 2011. “Terahertz spectroscopy andimaging-modern techniques and applications”. Laser Photon. Rev. 5,124-166; and Tonouchi, M., 2007. Cutting-edge terahertz technology. Nat.Photon. 1, 97-105, each incorporated herein by reference in theirentirety). Applying terahertz technology in the biomedical context hasinvoked considerable interest, as rich spectroscopic features, such ascollective structural vibrational modes of proteins and DNA are found toexist at these frequencies. (See Al-Naib, I., 2017. “Biomedical sensingwith conductively coupled terahertz metamaterial resonators”. IEEE J.Sel. Top. Quantum Electron. 23, 4700405; Baras, T., Kleine-Ostmann, T.,Koch, M., 2003. “On-chip THz detection of biomaterials: a numericalstudy”. J. Biol. Phys. 29, 187-194; Markelz, A. G., 2008. “Terahertzdielectric sensitivity to biomolecular structure and function”. IEEE J.Sel. Top. Quantum Electron. 14, 180-190, each incorporated herein byreference in their entirety).

Further, label-free identification of various biomedical analytes withterahertz waves has been proposed. (See Nagel, M., Richter, F.,Haring-Bolivar, P., Kurz, H., 2003. “A functionalized THz sensor formarker-free DNA analysis”. Phys. Med. Biol. 48, 3625-3630; and O'Hara,J. F., Withayachumnankul, W., Al-Naib, I., 2012. “A review on thin-filmsensing with terahertz waves”. J. Infrared Millimeter Terahertz Waves33, 245-291, each incorporated herein by reference in their entirety).This technique improved over previous testing methods, in which theunidentified analyte was labeled with fluorescent molecules. Apart fromrequiring an additional preparation step, which restricted the speed-and cost-efficiency, the analyte conformation may be altered in thelabeling process, lowering the yield and reliability of the method. (SeeFischer, B. M., Walther, M., Uhd Jepsen, P., 2002. “Far-infraredvibrational modes of DNA components studied by terahertz time-domainspectroscopy”. Phys. Med. Biol. 47, 3807-3814; and Mickan, S. P.,Abbott, D., Munch, J., Zhang, X.-C., 2002. “Noise reduction in terahertzthin film measurement using a double modulated differential technique”,Fluct. Noise Lett. 2, R13-R28, each incorporated herein by reference inits entirety).

Realizing the potential of label-free tests at terahertz frequencies hasbeen a challenging task due to the large difference between the sensingwavelength (0.3 mm at 1 Thz) when it is compared with the thickness of atiny quantity of analyte (in nanometers). In order to access the desiredinformation, thin-film sensors with very high sensitivity must bedeveloped. There have been several approaches introduced in recentyears, but the various techniques suffer from limitations, such as lowfrequency response, thickness of films, low quality-factor, etc. (SeeAl-Naib, I., Withayachumnankul, W., 2017. “Recent progress in terahertzmetasurfaces”. J. Infrared Millimeter Terahertz Waves 38, 1067-1084;Gupta, M., Srivastava, Y. K., Manjappa, M Singh, R., 2017. “Sensing withtoroidal metamaterial”. Appl. Phys. Lett. 110, 121108; O'Hara, J. F.,Singh, R., Brener, I., Smirnova, E., Han, J., Taylor, A. J., Zhang, W.,2008. “Thin-film sensing with planar terahertz metamaterials:sensitivity and limitations”. Opt. Express 16, 1786-1795; and O'Hara, “Areview on thin-film sensing with terahertz waves” (2012); andWithayachumnankul, W., O'Hara, J. F., Cao, W., Al-Naib, I., Zhang, W.,2014. “Limitation in thin-film sensing with transmission-mode terahertztime-domain spectroscopy”. Opt. Express 22, 972, each incorporatedherein by reference in their entirety).

For thin-film sensors to function efficiently, the frequency responsemust show a sharp transition to allow the recognition of small changesin the frequency response due to the modification in the dielectricenvironment. Conventionally, the steepness of this transition has beenconsidered as a direct measure of the sensor sensitivity. (See Singh,R., Cao, W., Al-Naib, I., Cong, L., Withayachumnankul, W., Zhang, W.,2014. “Ultrasensitive terahertz sensing with high-Q Fano resonances inmetasurfaces”. Appl. Phys. Lett. 105, 171101, incorporated herein byreference in its entirety).

Planar metamaterials or metasurfaces can be considered as filters ofelectromagnetic waves and consist of two-dimensional arrays of identicalmetallic resonators. The metamaterials are formed as a rectangular orcircular ring with a gap. When THz radiation is applied to the sensor,an electric field develops within the gap. This field can be detected byspectral imaging. (See Al-Naib, I., Jansen, C., Singh, R., Walther, M.,Koch, M., 2013. “Novel THz metamaterial designs: from near- andfar-field coupling to high-Q resonances”. IEEE Trans. Terahertz Sci.Technol. 3, 772-782; Fedotov, V. A., Rose, M., Prosvirnin, S. L.,Papasimakis, N., Zheludev, N. I., 2007. “Sharp trapped-mode resonancesin planar metamaterials with a broken structural symmetry”. Phys. Rev.Lett. 99, 147401, each incorporated herein by reference in theirentirety).

A drawback of traditional designs is the strong free-space coupling ofthe structural elements, which induces high radiation losses, resultingin lower quality (Q-) factors of the devices. Different designs havebeen investigated in order to suppress the radiation losses, and oneapproach utilized the asymmetric sub-radiant Fano resonance excited bybreaking the symmetry of a double-split ring resonator. (See Fedotov etal. (2007); and Singh, R., Al-Naib, I. A. I., Koch, M., Zhang, W.,2011a. “Sharp Fano resonances in THz metamaterials. Opt. Express” 19,6312-6319; Al-Naib, I., Hebestreit, E., Rockstuhl, C., Lederer, F.,Christodoulides, D Ozaki, T., Morandotti, R., 2014. “Conductive couplingof split ring resonators: a path to THz metamaterials with ultrasharpresonances”. Phys. Rev. Lett. 112, 183903; Singh, R., Al-Naib, I. A. I.,Yang, Y., Roy Chowdhury, D., Cao, W., Rockstuhl, C., Ozaki, T.,Morandotti, R., Zhang, W., 2011b. “Observing metamaterial inducedtransparency in individual Fano resonators with broken symmetry”. Appl.Phys. Lett. 99, 201107, each incorporated herein by reference in theirentirety).

Sharp Fano resonances have been found to be excited by means ofmetasurfaces consist of asymmetric split-rectangular resonators (ASRs).After applying the analyte onto the sensor surface, the resonancered-shifted due to the dielectric environment alteration. This shift ofthe resonance frequency was considered as a measure of the refractiveindex, i.e. the analyte type and its thickness. After measuring thefrequency response, the results were normalized to the frequencyresponse of a bare substrate. Measurable results were achieved when theanalyte thin-film refractive index was 1.6 with a thickness of onemicron or larger. For instance, a frequency shift of 10 GHz at aresonance frequency of 0.52 THz was achieved when the analyte thicknesswas one micron and the shift increased to 29 GHz at analyte thickness of16 μm where it is eventually saturated. In order to enable sensing ofanalytes with a thickness less than one micron, there have been attemptsto address this problem by using membranes or a very low dielectricconstant substrate in reflection mode. (See Chen, Y., Al-Naib, I. A. I.,Gu, J., Wang, M., Ozaki, T., Morandotti, R., Zhang, W 2012. “Membranemetamaterial resonators with a sharp resonance: a comprehensive studytowards practical terahertz filters and sensors”. AIP Adv. 2, 22109;Tao, H., Strikwerda, A. C., Liu, M., Mondia, J. P., Ekmekci, E., Fan,K., Kaplan, D. L., Padilla, W. J., Zhang, X., Averitt, R. D., Omenetto,F. G., 2010. “Performance enhancement of terahertz metamaterials onultrathin substrates for sensing applications”. Appl. Phys. Lett. 97,261909; and Reinhard, B., Schmitt, K. M., Neu, J., Beigang, R. R., Rahm,M., Wollrab, V., 2012. “Metamaterial near-field sensor fordeep-subwavelength thickness measurements and sensitive refractometry inthe terahertz frequency range”. Appl. Phys. Lett. 100, 221101; Yu etal., “The potential of terahertz imaging for cancer diagnosis: A reviewof investigations to date”, Quant Imaging Med Surg. 2012 March; 2(1):33-45, each incorporated herein by reference in their entirety).

However, these extra preparation conditions hinder the achievement ofhigh throughput thin-film sensors. Moreover, it is highly desirable tohave a fast process for the whole sensing procedure. Hence, themeasurement scan time should be minimized as much as possible. Currentpractices use long scan times carried out by the THz spectrometers.These long scan times have been considered essential to achieving therequired resolution in the frequency domain in order to discern closepoints. For instance, a 200 ps scan time is required to achieve 5 GHzresolution in the frequency domain.

Further, it would be desirable to utilize standard photolithographyalong with transmission mode spectroscopy to enable the discernment ofthe resonance shift when the analyte thickness is less than one micronat a reasonable scan time.

Aspects of the present disclosure present a solution to the problemspresented above by a method of biosensing analysis including anamplitude difference referencing technique (ADRT), which allowsdetection of resonance shifts for analyte thicknesses of 0 to 2 μm.Further, a new biosensor probe is presented which provides comparableresults with only about 20% of the amount of biomaterial needed foranalysis using conventional methods.

SUMMARY

In an exemplary embodiment, a system for biomolecule identification byterahertz sensing, comprising an asymmetric triple split-rectangular(ATSR) metamaterial biosensor having a metamaterial structure formed ona substrate, the metamaterial structure including three mutually opposedgaps. The structure includes a receiving region for an analyte to bedeposited on the metamaterial structure. The system includes a terahertzradiation source having a range of frequencies for interrogating theasymmetric triple split-rectangular metamaterial biosensor and aterahertz receiver for receiving electrical signals that are modified bythe analyte at the gaps. A database record stores a list of resonantfrequencies for a plurality of different analyte types. A controller hascircuitry configured to cause the terahertz radiation source to projectterahertz radiation at a range of frequencies onto the unknown analyte,receive the terahertz frequency response from the receiver, anddetermine the resonant frequency from the frequency responses of theasymmetric triple split-rectangular metamaterial biosensor by anamplitude difference referencing technique, and match the resonantfrequency to the database record to identify the analyte type.

In another exemplary embodiment, an asymmetric triple split-rectangular(ATSR) metamaterial biosensor is disclosed, comprising a first E shapedsensor part deposited upon a substrate, the first E shaped sensor havingthree evenly spaced legs each of length l; a second inverted E shapedsensor part deposited upon the substrate, the second inverted E shapedsensor part having three evenly spaced legs each of length k, where l isgreater than k and the sum of l and k is less than the width of thebiosensor. Each leg of the first E shaped sensor part includes a firstend connected at a right angle to a back of the E shape and a second endhaving two perpendicular extensions in the plane of the sensor. Each legof the second inverted E shaped sensor part includes a third endconnected at a right angle to a back of the inverted E shape and afourth end having two perpendicular extensions in the plane of thesensor. The extension end of each leg of the E shaped sensor mutuallyopposes the extension end of a corresponding leg of the inverted Eshaped sensor; and each extension end of the E shaped sensor isseparated from the corresponding extension end of the inverted E shapedsensor by a gap.

In another exemplary embodiment, a method for biomolecule identificationby terahertz sensing is described, comprising transmitting, by aterahertz light source, a terahertz wave in a range of frequencies to anasymmetric triple split-rectangular metamaterial biosensor which may beloaded with an analyte, receiving, by a terahertz receiver, a frequencyresponse from the biosensor; transmitting the frequency response fromthe terahertz receiver to a sensor control system, wherein the sensorcontrol system includes a controller having processing circuitryconfigured for analyzing the frequency response by an amplitudedifference referencing technique to determine a resonant frequency; andidentifying the analyte type by matching the resonant frequency to adatabase record.

The foregoing general description of the illustrative embodiments andthe following detailed description thereof are merely exemplary aspectsof the teachings of this disclosure, and are not restrictive.

BRIEF DESCRIPTION OF THE DRAWINGS

A more complete appreciation of this disclosure and many of theattendant advantages thereof will be readily obtained as the samebecomes better understood by reference to the following detaileddescription when considered in connection with the accompanyingdrawings, wherein:

FIG. 1A illustrates a schematic of a conventional metasurface unit cell.

FIG. 1B illustrates an asymmetric double split-rectangular (ASR)metamaterial unit cell with detailed geometric dimensions.

FIG. 2A is a graph illustrating the transmission amplitude spectra ofthe uncoated and coated metasurface with 0.25 μm thick analyte using theconventional normalization method.

FIG. 2B illustrates the amplitude difference referencing technique usedwith the uncoated metasurface response as a reference.

FIG. 3A is a graph illustrating the transmission amplitude spectra usingthe conventional testing method for different submicron thickness valuesof analyte deposited on the metasurface.

FIG. 3B is a graph illustrating the amplitude difference referencingtechnique for different submicron thickness values of analyte depositedon the metasurface.

FIGS. 4A and 4B are graphs illustrating (A) Fano resonance frequencyshift and (B) peak-to-peak amplitude difference versus analytethickness.

FIG. 5 illustrates an asymmetric triple split-rectangular (ATSR)metamaterial unit cell.

FIG. 6 is the asymmetric triple split-rectangular (ATSR) metamaterialunit showing the position at which the analyte is applied.

FIG. 7A shows the transmission amplitude frequency response for theATSR.

FIG. 7B shows the amplitude difference for the ATSR.

FIG. 8A illustrates the mean refractive indices of tissue samples.

FIG. 8B illustrates a CW THz laser system which may be used to performthe exemplary sensing.

FIG. 9 depicts the terahertz measurement system.

DETAILED DESCRIPTION

In the drawings, like reference numerals designate identical orcorresponding parts throughout the several views. Further, as usedherein, the words “a,” “an” and the like generally carry a meaning of“one or more,” unless stated otherwise. The drawings are generally drawnto scale unless specified otherwise or illustrating schematic structuresor flowcharts.

Furthermore, the terms “approximately,” “approximate,” “about,” andsimilar terms generally refer to ranges that include the identifiedvalue within a margin of 20%, 10%, or preferably 5%, and any valuesthere between.

Terahertz radiation has frequencies of 0.1 to 30 terahertz on thespectral scale. One terahertz is 10¹² Hz or 1000 GHz. Wavelengths ofradiation in the terahertz band correspondingly range from 1 mm to 100μm. The terahertz region lies between the microwave and infrared regionsof the electromagnetic spectrum and is strongly attenuated by water andvery sensitive to water content. The presence of cancer often causesincreased blood supply to affected tissues and the increase in tissuewater content acts as a natural contrast mechanism for terahertz imagingof cancer. The increased water content increases the refractive index ofthe biomaterial and influences the resonant frequency of measurements.Because of these characteristic properties, terahertz imaging forbiological applications and terahertz spectra is a valuable tool foridentifying biomolecules, including cancer cells.

The top surface of the metamaterial is covered with a thin layer ofanalyte in order to examine various sensing parameters. The sensitivityand corresponding figure of merit (FoM) of the odd and even resonantmodes are analyzed with respect to coated analyte films. In anon-limiting example, the analyte may be a thin film with a coating of acancer biomolecule. In a further non-limiting example, the analyte maybe a film coated with blood containing AIDS biomolecules. Alternatively,the analyte may be a layer of biomolecule material placed upon thesensing domain of the sensor and does not need to be placed on a filmcarrier.

When an unidentified biomolecule analyte has sub-micron thickness, thesample volume is very small, resulting in a very low amplitude signalresponse, therefore the identification of sub-micron thicknessbiomolecules is a challenging task. Therefore, a sensor known as anasymmetric split-rectangular (ASR) metamaterial biosensor (referred toas the conventional sensor in the present disclosure, see FIG. 1A),having gold electrodes separated by gaps has been used to enhance thefrequency response. Upon applying an analyte to the sensor surface, theresonant frequency is red-shifted due to the dielectric environmentalteration. This shift of the resonance frequency is considered as ameasure of the refractive index, which is related to the analyte typeand its thickness. The amplitude of the frequency response is very smalland previous methods of analysis have suffered in accurateidentification of samples less than 2 μm in thickness. Further, thisbiosensor is not able to accurately measure the frequency response ofvery small samples of analyte less than 2 μm in thickness.

Aspects of this disclosure are directed to a system for biomoleculeidentification by terahertz sensing, an asymmetric triplesplit-rectangular (ATSR) metamaterial biosensor, and method forbiomolecule identification by terahertz sensing.

In the first aspect, a system for biomolecule detection by terahertzsensing is described. The system uses an asymmetric triplesplit-rectangular (ATSR) metamaterial biosensor upon which a sub-micronthickness analyte is placed. The resonant response is analyzed by anamplitude difference referencing technique (ADRT). Correlation of theresonant frequency against the refractive indices of correspondingbiomolecules of the same thickness identifies a particular biomolecule,such as a cancer cell.

In a further aspect, the present disclosure describes a system for theevaluation of thin-films in semiconductor fabrication processes byterahertz sensing.

In one aspect, the conventional sensing cell as shown in FIG. 1A issubjected to a terahertz radiation and the ADRT is used to measure theresponse.

In another aspect, the asymmetric triple split-rectangular (ATSR)metamaterial biosensor of the present disclosure is subjected toterahertz radiation and the ADRT is used to measure the response.

In an aspect, a method for biomolecule detection includes using anamplitude difference referencing technique (ADRT) to determine thefrequency response by interrogating the analyte covered surface of anASR or an ATSR with terahertz radiation. An uncoated metamaterial sensorprovides a reference frequency response. The sensitivity level using theconventional method of normalization by dividing an analyte coatedmetasurface transmission amplitude response by its bare substratecounterpart response is compared to the sensitivity level using theADRT. The ADRT is determined by subtracting the frequency response ofthe coated metasurface from the frequency response of the uncoatedmetasurface structure.

The conventional metasurface unit cell consists of double-splitrectangular asymmetric metal resonators 106 deposited on top of adielectric substrate 104. As shown in FIG. 1A, a 3D view of theasymmetric double split-rectangular (ASR) metamaterial unit cell isshown with an analyte layer applied to the metasurface sensor. Theanalyte 102 is applied to the metallized side and covers the entiremetallized area. FIG. 1B shows the geometrical dimensions of theasymmetric split-rectangular resonator unit cell. In a non-limitingexample, the geometrical dimensions of the resonator used for simulationare: a side length of l=80 μm, a microstrip line width of w=10 μm, a gapof g=5 μm, an asymmetry distance from the center line of d=5 μm, and aperiodicity of p=100 μm. The refractive index of the high resistivitysilicon substrate wafer is 3.42. The thickness of the gold metallizedmetamaterial resonators is 200 nm from the top of the silicon substrate.Alternatively, the substrate may be a sapphire wafer.

Each resonator in the metamaterial is composed of a metallic loop with asplit. Upon excitation by an incident terahertz wave, this resonatorexhibits a Lorentzian resonance response. This response is analogous tothat obtainable from a lumped RLC circuit with capacitance C andinductance L approximately determined by the properties of thedielectric gap and the metallic loop, respectively. Upon resonance, acollection of charge is strongly established at the dielectric gap,which results in the oscillating current in the loop and the strongelectric field across the gap. This confined electric field is highlysensitive to a change in the surrounding material. Samples that areplaced in the modify the capacitance of the ring, causing a shift in theresonance frequency. Since the gap region is very small and themetamaterial structure is highly resonant, only small sample amounts arenecessary to invoke a measurable change.

Simulations were conducted using frequency domain solver simulationsoftware. In a non-limiting example, the frequency domain solversimulation software is the Computer Simulation Technology (CST)Microwave Studio by Dassault Systemes, 175 Wyman St., Earth Building,Waltham, Mass. 02451, U.S., https://www.cst.com/products/cstmws,incorporated herein by reference in its entirety), which is based on thefinite integration technique. Periodic boundary conditions have beenutilized to mimic the actual configuration and normal incidence planewave excitation has been applied. Since the electric field is orientedperpendicular to the two gaps, as indicated in the inset of FIG. 1B, anasymmetric sharp Fano resonance as well as a symmetric dipole resonanceare excited.

In physics, a Fano resonance is a type of resonant scattering phenomenonthat gives rise to an asymmetric line-shape. Interference between abackground and a resonant scattering process produces the asymmetricline-shape.

Conventionally, three sets of data are measured in the time-domain for:(i) a bare substrate, (ii) uncoated metasurface structure, and a (iii)coated metasurface after depositing an analyte on top of the metasurfacestructure. After converting the measured data to the frequency domain,the latter two responses are normalized to the response of the baresubstrate.

FIG. 2A shows an example of using the conventional analysis method todetermine the normalized uncoated metasurface frequency response (solidline 208). The Fano asymmetric resonance is at 0.396 THz and the dipoleresonance is at 0.7 THz (not shown). The spectral response of the Fanoresonance features a small bandwidth of 34 GHz and therefore the qualityfactor (defined as resonance frequency/bandwidth) of this resonance isalmost 12 for the given configuration. After an analyte thickness of0.25 μm is applied on top of the metasurface, the frequency response(dotted line 210) normalized to the bare substrate is red-shifted by 1GHz to 0.395 THz as shown in FIG. 2A. This red-shift in the frequencyresponse is a consequence of the alteration in the dielectricenvironment of the resonators. The shift/refractive index unit is 1GHz/(1.6-1.0)=1.67 GHz/RIU (refractive index unit), which represents avery low sensitivity. As shown in FIG. 2A, this difference is notmeasurable with any practical degree of accuracy, thus cannot be used tomeasure submicron analyte thicknesses. Further, it is important to notethat this very small red-shift requires a minimum of 1000 ps scan timemeasurements in order to discern the coated sample response from theuncoated one.

However, the amplitude difference referencing technique (ADRT) of thepresent disclosure may be used to resolve the frequencies to a highdegree of accuracy. In the amplitude difference referencing techniquethe frequency response of uncoated metasurface is subtracted from thefrequency response of the coated metasurface without the normalizationused in the conventional method. The result (line 212) exhibits a veryclear amplitude difference signature as shown in FIG. 2B. Thepeak-to-peak difference is 8.6%, which can easily be measured anddifferentiated from the noise floor using either conventional terahertztime-domain spectrometers or state-of-the-art systems with a dynamicrange of 90 dB. (See Vieweg, N., Rettich, F., Deninger, A., Roehle, H.,Dietz, R., Gabel, T., Schell, M., 2014. “Terahertz-time domainspectrometer with 90 dB peak dynamic range”. J. Infrared MillimeterTerahertz Waves 35, 823-832, incorporated herein by reference in itsentirety). This result confirms that the amplitude differencereferencing technique is able to detect the frequency responses ofbiomolecules using analyte thicknesses is in the range of sub-microns.

In order to further evaluate the amplitude difference referencingtechnique, transmission amplitude spectra for analyte thicknesses of0.25, 0.5, 1, and 2 μm are compared between the conventionalnormalization method (FIG. 3A(a-d)) and the ADRT (FIG. 3B(e-h)). Thevertical dashed line 314 shows the location of the Fano resonance dipfor the uncoated metasurface, i.e. without the analyte. FIG. 3A(a-d))show the normalized transmission amplitude where the small red-shiftincreases with the increase in the analyte thickness. The correspondingred-shift in the frequency response is 1, 2, 6, and 9 GHz, respectively,which results are very hard to determine accurately. For a largeranalyte thicknesses of 5 μm, 10 μm, and 15 μm (not shown), the red-shiftincreased to 14.5, 18.6, and 20.1 GHz, respectively. Although theconventional normalization method performs poorly below 2 μm, it may beuseful for analyte thickness of 2 μm or more. On the contrary, the ADRTshows a large amplitude difference for the same range of the analytethickness as shown in FIG. 3(e)-(h) ranging from 8.6% to 55.8% foranalyte thicknesses of 0.25 μm to 2 μm, respectively, which results areeasily detectable and measurable.

This large amplitude difference represents the significant steepness ofthe flank of the amplitude response. For instance, the steepness (thefrequency derivative) of the amplitude response of the uncoated sampleis 6.3% per GHz (see FIG. 2B), which results in an amplitude differenceof 37% for an analyte thickness of 1 μm as shown in FIG. 3B(g).Therefore, decreasing the steepness of the amplitude response leads to adecrease in the amplitude difference. This can be demonstrated byincreasing the asymmetry distance from the center line, d, of theasymmetric split-rectangular resonator to be 20 μm instead of 5 μm. Inthis case, the steepness of the amplitude frequency response decreasesto 1.6% per GHz. After analyzing the amplitude response of the coatedsample with 1 μm analyte, the amplitude difference was found to be 18%.

The performance of the conventional normalization method versus the ADRTfor a wide range of the analyte thickness between 0.25 μm and 15 μm isshown in FIGS. 4A and 4B. FIG. 4A shows the resonance frequency shiftand the peak-to-peak amplitude difference using the conventionalnormalization method and FIG. 4B shows the results found using theamplitude difference referencing technique, both as a function of theanalyte thickness, respectively. The relationship between the resonancefrequency red-shift and the analyte thickness is exponential for smallthicknesses less than 5 μm and gradually reaches a saturated value asrevealed in FIG. 4A.

In contrast, saturation using the ADRT takes place for analytethicknesses greater than 2 μm as shown in FIG. 4B. This saturation isattributed to the fact that the amplitude difference value cannot bemore than the difference between the peak and the dip of the amplituderesponse near resonance. It is evident from these results that the ADRTadvantageously resolves frequency response for sub-micron analytethickness below 2 μm.

TABLE I Raw data of FIG. 4B Analyte Shift Peak-to-peak Thickness in GHzAmplitude Difference 0.25 1 8.6 0.5 2.3 20.7 1 6 37 2 8.8 55 5 14.4558.9 10 18.6 59 15 20.1 59

It is clear that the shift in GHz is very small when the analytethickness is sub-micron using the conventional method. However, thepeak-to-peak amplitude difference is significant and can be easilyevaluated.

The aspect of the present disclosure describing an improved biosensordesign is shown with respect to FIG. 5 . The asymmetric triplesplit-rectangular (ATSR) metamaterial biosensor improves the confinementof the spatial electric field over the sensor design of FIG. 1B. Thissensor is deposited by photolithography upon a high resistivity siliconsubstrate wafer and the metalized metamaterial resonators may be made ofgold, similarly to the conventional sensor cell of FIG. 1A. Thebiosensor has two parts, 530 and 532. Each part has three sensor legs(516, 518, 520) shown on the left of FIG. 5 and three sensor legs (534,536, 538) shown on the right. The left shape is an E shape and the rightshape is an inverted E shape. The sensor legs are mutually opposing witha small gap between their ends. In a non-limiting example and purely forexplanatory purposes, the sensor legs (516, 518, 520) are each about 10μm in width, and each leg ends in a probe (522, 524, 526) havingextensions of 7.5 μm on either side of the width of the probe at itsrespective gap region. These additional extensions help to confine theelectric field between the probes. The width of each gap is about 2 μm.In a non-limiting example, the length of legs 516, 518, 520=32.5 μm, thelength of legs 534, 536, 538=22.5 μm, the width of each leg=10 μm, theperiodicity=100 μm and the distance between the legs may be 2 μm(asymmetry distance from the center line of d=5 μm). As shown in theFIG. 6 , it is not necessary to cover the entire area of the sensor withthe analyte, but only the parts of the sensor near the gaps. In FIG. 6 ,about 20% of the sensor area has been coated with the analyte. In anon-limiting example, the width 628 of the analyte is about 20 μm andits thickness is 1 μm. Thus, only one fifth of the amount of analytematerial used by the conventional sensor is needed to achieve anapproximately 34% amplitude difference as opposed to the 37% amplitudedifference using the asymmetric double split-rectangular (ASR)metamaterial sensor. This is an important achievement, as often only atiny amount of analyte may be available for testing after extractionfrom cancerous cells or biomolecules. The substrate may further includea raised ridge (not shown) around the first and second part to receivethe analyte. A raised ridge may be formed of silicon dioxide or otherinert material. Alternatively, a microfluidic channel may be employed tocontain the analyte on the metamaterial. The analyte may be dropped ontothe metamaterial structure, pumped, pipetted, applied in sheet form, orany other method of application as is conventionally known. Amicrofluidic pumping method of applying analyte may be used. (See Genget al., “A Route to Terahertz Metamaterial Biosensor Integrated withMicrofuidics for Liver Cancer Biomarker Testing in Early Stage”,Scientific Reports, Vol. 7, pp. 1-11,https://www.nature.com/articles/s41598-017-16762-y.pdf, incorporatedherein by reference in its entirety).

FIG. 7A, 7B illustrate a simulation using both the conventionalnormalization method with the ATSR and the ADRT with the ATSR. FIG. 7Ashows that the using the ATSR provides a clearer frequency separationthan the conventional normalization method. Further, ilt is evident fromFIG. 7B that the peak-to-peak amplitude difference using the ATSR andthe ADRT with only 20% of the metastructural surface covered is almost34%, while it was 37% when using the ADRT with conventional probe (seeFIG. 3B(g) with the entire metastructural surface structure was coveredwith the analyte. In summary, the asymmetric triple split-rectangular(ATSR) metamaterial biosensor with only 20% of the surface area coveredwith analyte performs comparably to the conventional biosensor fullycovered with analyte. This result is of great benefit in analysis ofbiomolecules as only one-fifth as much biomaterial must be taken fromthe patient in a biopsy or other analysis procedure.

In conclusion, the biosensor design of FIG. 6 offers a better electricfield confinement compared to conventional designs. Moreover, only 20%as much analyte is needed to achieve similar results. Further, the ADRTanalysis method yields much better resolution for analyte thicknessesbelow 2 μm.

As the refractive index of different biomolecule analytes ranges from1.4 to 1.6 in DNA and 1.6 to 2.0 in RNA, an average value of the analyterefractive index of 1.6 was chosen for the purpose of the analysis. (SeeYahiaoui, R., Strikwerda, A. C., Jepsen, P. U., 2016. “Terahertzplasmonic structure with enhanced sensing capabilities”. IEEE Sens. J.16, 2484-2488, incorporated herein by reference in its entirety). In theconventional normalization method, the refractive index may bedetermined by measuring the resonant frequency shift of a knownthickness of analyte. The refractive index may be referenced to a tableor database of lookup values to identify the analyte. When the sample isthinner than one micron, the sensitivity will be very low as shown inFIG. 3A and may lead to an incorrect identification of the analyte.Conversely, the amplitude difference referencing technique (ADRT) usesthe amplitude difference and it is evident from the results shown inFIG. 3B and FIG. 7(b) that it is far more sensitive to a small variationin refractive index when the analyte thickness is less than two micron.

FIG. 8A depicts the refractive indices of fat, liver and kidney tissuesamples versus frequencies ranging from 0.25 to 1.5 THz. This graphshows that the refractive index may be used to identify the type ofcell. (See Yu et al., “The potential of terahertz imaging for cancerdiagnosis: A review of investigations to date”, Quant Imaging Med Surg.2012 March; 2(1): 33-45, incorporated herein by reference in itsentirety).

Further, it is noted that tryptophan is a good biomarker for skin tumordiagnosis. Tryptophan has resonant absorptions at 1.42 and 1.84 THz.These resonances may be used to identify skin cancer cells by using theasymmetric triple split-rectangular (ATSR) metamaterial biosensor withthe amplitude difference referencing technique (ADRT).

Terahertz time domain spectroscopy (THz-TDS) has been conventionallyused to take the measurements. THz-TDS offers the complete time domainresponse in one shot, i.e. the entire frequency response from themeasured time-domain response may be determined. The measurement time ison the order of 10-20 minutes, depending on the required resolution.

In a non-limiting example, the THz-TDS device may be the TERA K15, Allfiber-coupled Terahertz Spectrometer, manufactured by Menlo SystemsInc., 56 Sparta Avenue, Newton, N.J. 07860, US,https://www.menlosystems.com/products/thz-time-domain-solutions/all-fiber-coupled-terahertz-spectrometer.

However, as shown in FIG. 3(e)-(h), a complete frequency response is notneeded to calculate the amplitude difference as the peak (using arefractive index of 1.6) localized around 0.4 THz. Hence, we can use THzcontinuous wave systems (THz-CW). These systems are much more affordablethan their THz-TDS counterparts as they rely on 1550 nm communicationlasers, which make the whole measurement system much more costeffective. Moreover, the THz-CW device is more compact and reliable. Ina non-limiting example, a CW THz laser may be the CW TILL Lasermanufactured by Sacher Lasertechnik, LLC, 5765 Equador Way, Buena Park,Calif. 90620, U.S.Ahttps://www.sacher-laser.com/home/scientific-lasers/thz_tera_hertz_generation/tera_hertz/tec_450_thz_generation.html.A CW THz laser system which may be used to interrogate the biosensor isshown in FIG. 8B.

The terahertz imaging system of the present disclosure is shown in FIG.9 . FIG. 9 depicts a terahertz source 960 which generates a teraherzwave 964 in a range of frequencies onto an asymmetric triplesplit-rectangular (ATSR) metamaterial biosensor 930 having an analyteapplied upon it. The biosensor responds with a set of electrical signals966 that are modified by the analyte at the gaps of the sensor which arereceived by the terahertz receiver 962. The terahertz receiver 962 isconnected to control system 940 having a controller 942, a processor 943connected to the controller, an ADRT module 944 and a database 946 whichholds a record of the relationship of an amplitude difference responseto a refractive index of a biomolecule and the biomolecule type. Thedatabase may also include frequency responses of biomolecules based onanalyte thickness. The processor 943 sends terahertz signals to the ADRTmodule for frequency analysis and references the database 946 toidentify the cell type. The identification is sent to the controller,which transmits data to an I/O port 948 for display (950), printing (notshown), and the like.

The first embodiment is illustrated with respect to FIGS. 1A, 5, 6 and 9. The first embodiment describes a system for biomolecule identificationby terahertz sensing, comprising an asymmetric triple split-rectangular(ATSR) metamaterial biosensor 500 having a substrate 104; a metamaterialstructure formed on the substrate, the metamaterial structure includingthree mutually opposed gaps 570 which form a sensing domain. An unknownanalyte 102 may be deposited on the metamaterial structure. The analytemay be a film, a film coated with a biomolecule sample, such as blood orbiopsy tissue, a thin layer of biomolecule dropped, pipetted, pumped orotherwise placed on the sensing domain, etc.

A terahertz radiation source 960 having a range of frequencies forinterrogating the asymmetric triple split-rectangular metamaterialbiosensor and a terahertz receiver 962 for receiving electrical signalsgenerated at the gaps are included in the system. The system furtherincludes a database 946 with records having a list of resonantfrequencies for a plurality of different analyte types and a controller942 having circuitry configured to cause the terahertz radiation source964 to project terahertz radiation at a range of frequencies onto theunknown analyte 102; receive the terahertz frequency response from thereceiver 962, and determine an amplitude difference from the frequencyresponses of the asymmetric triple split-rectangular metamaterialbiosensor by an amplitude difference referencing technique; and matchthe resonant frequency to the database record to identify the analyte orbiomolecule type.

The substrate 104 is one of a high resistivity silicon substrate waferor a sapphire wafer.

The metamaterial biosensor includes a first E shaped sensor part 530having three evenly spaced legs (516, 518, 520) each of length l and asecond inverted E shaped sensor part 532 having three evenly spaced legs(534, 536, 538) of length k, where l is greater than k and l and k areeach less than 80 μm. Each leg of the first E shaped sensor partincludes a first end connected at a right angle to a back of the E shapeand a second end having two perpendicular extensions (shown as 522, 524,526 on the second part) in the plane of the sensor; and wherein each legof the second inverted E shaped sensor part includes a third endconnected at a right angle to a back of the inverted E shape and afourth end having two perpendicular extensions (522, 524, 526) in theplane of the sensor.

The extension end of each leg of the E shaped sensor mutually opposesthe extension end of a corresponding leg of the inverted E shaped sensoras shown in FIG. 5 ; and each extension end of the E shaped sensor isseparated from the corresponding extension end of the inverted E shapedsensor by a gap 570.

The gaps confine the electric field between the extension ends and thefrequency response is measured at the gaps.

An analyte applied to the sensing domain may have a thickness in therange of 0.1 to 2 μm.

The metamaterial is gold.

Each gap 570 may have a width of 1-3 μm, preferably 2 μm.

The analyte is one of a cancer cell, a cancer biomarker and abiomolecule.

The amplitude difference of the resonant frequencies corresponds to therefractive index of the unknown analyte and the refractive index ismatched to the analyte type in the database record.

The second embodiment is illustrated with respect to FIGS. 1A, 5, 6 and9 . The second embodiment describes an asymmetric triplesplit-rectangular (ATSR) metamaterial biosensor, comprising a substrate104; a first E shaped sensor part 530 deposited upon the substrate, thefirst E shaped sensor having three evenly spaced legs (516, 518, 520)each of length l; a second inverted E shaped sensor part 532 depositedupon the substrate, the second inverted E shaped sensor part havingthree evenly spaced legs (534, 536, 538) each of length k, where l isgreater than k and the sum of l and k is less than 80 μm. Each leg ofthe first E shaped sensor part 530 includes a first end connected at aright angle to a back of the E shape and a second end having twoperpendicular extensions (522 a, 524 a, 526 a) in the plane of thesensor. Each leg of the second inverted E shaped sensor part 532includes a third end connected at a right angle to a back of theinverted E shape and a fourth end having two perpendicular extensions(522 b, 524 b, 526 b) in the plane of the sensor. The extension end ofeach leg of the E shaped sensor mutually opposes the extension end of acorresponding leg of the inverted E shaped sensor as shown in FIG. 5 andeach extension end of the E shaped sensor is separated from thecorresponding extension end of the inverted E shaped sensor by a gap570.

The metamaterial is gold. The gap may be 1-3 μm, preferably 2 μm.

The substrate is one of a high resistivity silicon substrate wafer or asapphire wafer.

The substrate further may include a raised ridge of height h whichsurrounds the first and second part, wherein the height h of the raisedridge is greater than a thickness of the metamaterial.

Alternatively, the substrate may include a microfluidic channelconfigured for receiving analyte pumped into the channel.

The third embodiment is illustrated by FIGS. 1A, 5, 6 and 9 . The thirdembodiment describes a method for biomolecule identification byterahertz sensing, comprising transmitting, by a terahertz source 960, aterahertz wave in a range of frequencies to an asymmetric triplesplit-rectangular metamaterial biosensor 500 loaded with an analyte 102;receiving, by a terahertz receiver 962, a frequency response from thebiosensor; transmitting the frequency response from the terahertzreceiver to a sensor control system 940, wherein the sensor controlsystem includes a controller 942 having processing circuitry 943configured for analyzing the frequency response by an amplitudedifference referencing technique 944 to determine an amplitudedifference percentage; and identifying the analyte type by matching theamplitude difference percentage to a database 946 record.

Analyzing the amplitude difference percentage by the amplitudedifference referencing technique comprises measuring the amplitude ofthe resonant frequency response of an unloaded substrate, measuring theamplitude of the resonant frequency response of the loaded substrate andsubtracting the amplitude of the resonant frequency response of theloaded substrate from the resonant frequency response of an unloadedsubstrate.

Identifying the analyte type further comprises accessing a databaserecord corresponding to the amplitude difference percentage, matchingthe amplitude difference percentage to a list of refractive indiceswhich correspond to known analyte types, and identifying the analytetype.

For the processing circuitry, Matlab was used to post-process the data.However, for a stand-alone compact system, a Raspberry Pi single boardcomputer or other computer system may be used for this purpose. TheRaspberry pi single board computer has been used in previous sensorsystems, but not in the context of biosensing. (See Gente et al.,“Outdoor Measurements of Leaf Water Content Using THz Quasi Time-DomainSpectroscopy”, J Infrared Milli Terahz Waves, 17 Jul. 2018,http://www.thz.org.mx/pdfs/outdoor.pdf, incorporated herein by referencein its entirety).

The present disclosure combines the ADRT testing method with a new probesensor design. Incorporation with a THz-CW compact system, and postprocessing with a Raspberry Pi single board computer provides a highlyeffective THz biosensing product capable of detecting very small amountsof biomolecules.

In summary, the amplitude difference referencing technique has beenevaluated in the present disclosure in order to sense unknown analyteswith sub-micron thickness. High Q-factor Fano resonance excited via theasymmetric split ring metamaterial resonators of the conventional sensorhas been utilized with the evaluation process of the ADRT and comparedto the conventional normalization method. Further, an asymmetric triplesplit-rectangular (ATSR) metamaterial biosensor was evaluated in whichonly 20% of the analyte was needed to yield comparable results withthose of a conventional sensor. The performance of the ATSR wasevaluated for a range of analyte thicknesses. The amplitude differenceachievement is impressive, considering that only sub-micron analytethickness have been applied to the metamaterial sensor. In the future,the ATSR used with the ADRT can be utilized to identify analytes withsub-micron thickness and hence pave the way for a new generation oflabel-free biomedical sensors.

Obviously, numerous modifications and variations of the presentinvention are possible in light of the above teachings. It is thereforeto be understood that within the scope of the appended claims, theinvention may be practiced otherwise than as specifically describedherein.

The invention claimed is:
 1. A method for identifying a biomolecule byterahertz sensing with a sensor control system for biomoleculeidentification, comprising: transmitting, by a terahertz radiationsource, a terahertz wave in a range of frequencies to a sensing domainof an asymmetric triple split-rectangular metamaterial biosensor;receiving, by a terahertz receiver, a Fano resonance frequency responsefrom the biosensor; transmitting the frequency response from theterahertz receiver to the sensor control system, wherein the sensorcontrol system includes a controller having processing circuitryconfigured for analyzing the frequency response by an amplitudedifference referencing technique; and identifying the biomolecule bymatching the amplitude difference to a database record, wherein thesensor control system, comprises: an asymmetric triple split-rectangular(ATSR) metamaterial biosensor having a substrate; a metamaterialstructure formed on the substrate, the metamaterial structure includingthree mutually opposed gaps which form the sensing domain, the gapsformed by: a first E shaped sensor part having three evenly spaced legseach of length l and a second E shaped sensor part having three evenlyspaced legs of length l, each leg having a length k, where l is greaterthan k and l and k are each less than 80 μm, wherein the first E shapedsensor part and the second E shaped sensor part are coplanar in theplane of the sensor such that the legs of the first E shaped sensor partand the legs of the second E shaped sensor part oppose one another andthe ends of the legs of the first E shaped sensor part are separatedfrom ends of the legs of the second E shaped sensor part by gaps; theterahertz radiation source having the range of frequencies forinterrogating the sensing domain; the terahertz receiver for receivingelectrical signals from the sensing domain; the database record having alist of amplitude differences for a plurality of different biomoleculeanalytes; a controller having circuitry configured to cause theterahertz radiation source to project terahertz radiation at a range offrequencies onto the sensing domain; receive the terahertz frequencyresponse from the receiver, and determine the amplitude difference fromthe frequency responses of the asymmetric triple split-rectangularmetamaterial biosensor by an amplitude difference referencing technique;and match the amplitude difference to the database record to identifythe biomolecule.
 2. The method of claim 1, further comprising analyzingthe frequency response by the amplitude difference referencing techniqueby: measuring the amplitude of the resonant frequency response of anempty sensing domain; measuring the amplitude of the resonant frequencyresponse of a loaded sensing domain; and subtracting the amplitude ofthe resonant frequency response of the loaded sensing domain from theresonant frequency response of the empty sensing domain.
 3. The methodof claim 1, wherein identifying the biomolecule further comprisesaccessing a database record corresponding to the amplitude difference;matching the amplitude difference to a list of refractive indices whichcorrespond to known biomolecules; and identifying the biomolecule.